1. Field of the Invention
This invention relates to the adhesion of tissue, cell or other polynucleotide-containing samples to analytical elements or devices for nucleic acid hybridization assay testing. More particularly, it relates to the use of naturally occuring adhesive substances in solid state hybridization assay testing of such samples for clinical diagnostic or research purposes.
2. Brief Description of the Prior Art
Histology, cytology and related fields have long used stained or unstained tissue sections for a wide variety of educational, experimental and clinical applications. Both paraffin and frozen tissue sections or cell samples, such as from blood or cervical lavage, are attached onto glass slides for microscopic examination.
One important aspect of these procedures is the reliability of the adherence of nucleic acids, cells and tissue sections to the underlying slide or other surface on which the examination is performed. A recurring problem, particularly for in situ hybridization assays, has been failure of adherence of the cells or tissue section to the slide. Treatment with, for example, HCl, H.sub.2 O.sub.2, proteinase digestion, prehybridization and hybridization at elevated temperatures or with formamide all can cause cell or tissue release. The cell digestion by proteinase K as part of the sample preparation for in situ hybridization assays has amplified the problem, even when extremely dilute preparations of the proteinase are used. See, for example, Morley, et al., In Situ Localization of Amylase mRNA and Protein. An Investigation of Amylase Gene Activity In Normal Human Parotid Cells, J. Histochem. and Cytochem., 35:9-14(1987) and Brigati, et al., Detection of Viral Genomes In Cultured Cells and Paraffin-Embedded Tissue Sections Using Biotin-Labeled Hybridization Probes, Virology, 126:32-50(1983).
Some of nature's most powerful adhesives are produced by sessile intertidal marine invertebrates such as mussels and the efforts to correlate adhesiveness of Mytilus edulis through disc-tipped threads termed "byssus" with some chemical composition has been hindered by the extreme insolubility of the disc proteins. This adhesive disc has been referred to by histologists as the "phenol gland" because of its storage of aromatic or "polyphenolic protein".
Waite, et al., The Bioadhesive of Mytilus Byssus: A Protein Containing L-Dopa, Biochem. Biophys. Res. Comm., 96:1554-1561(1980) report here that the adhesive disc and the polyphenolic protein of Mytilus contain significant amounts of L-3,4-dihydroxyphenylalanine (L-DOPA). Extensive isolation procedures are reported. An aromatic compound "A" was detected in hydrolysates and was said to be identical with standard L-DOPA based on several analytical criteria. Although the authors acknowledge that the function of DOPA in the disc protein is not known, they raise the question of whether DOPA confers unique adhesive properties on the disc protein and/or contributes to the interaction of collagen and polyphenolic protein.
Waite, et al., Polyphenolic Substance of Mytilus edulis: Novel Adhesive Containing L-DOPA and Hydroxyproline, Science 212:1038-1040 (1981) report that the adhesive discs of Mytilus edulis are rich in the amino acid 3,4-dihydroxyphenylalanine (dopa). An acid-soluble protein was extracted and purified from the phenol gland located in the byssus-secreting foot of the animal. This protein was found to be highly basic and to contain large amounts of lysine, dopa, and 3- and 4-hydroxyproline (hyp). The authors conclude that this protein contributes to byssal adhesion.
Waite, Evidence for a Repeating 3,4-Dihydroxyphenylalanine-and Hydroxyproline-containing Decapeptide in the Adhesive Protein of the Mussel, Mytilus edulis L., J. Biol. Chemin., 258:2911-2915(1983). Here, treatment of the adhesive protein with clostridial collagenase was reported to reduce the molecular weight by less than 10%. The collagenase-resistant fragment was reported to contain most or all of the Hyp and Dopa. Trypsin treatment of the polyphenolic protein was reported to result in extensive degradation. The major tryptic peptide (80%) contains 10 amino acids including Hyp and Dopa and was shown by sequence analysis to be H.sub.2 N-Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Dopa-Lys-COOH.
When the organism is placed on glass, the attachment plaques are reported to exhibit a mean adhesive tensile strength of 10.sup.6 newton-meter.sup.-2. The substance in the plaque mediating adhesion between the collagenous threads of the disc and the substrate is stated to be the polyphenolic protein. The authors mention that polyphenolic protein has attracted attention as an adhesive since, unlike most synthetic adhesives, its performance, polymerization, and longevity are not adversely affected by the presence of water. Although the reason for this resistance to water is reported to be unknown, it is speculated as likely related to the unusual composition of the polyphenolic protein.
Waite, U.S. Pat. No. 4,496,397 discloses a method for purifying polyphenolic proteins rich in catechol, such as those from Mytilus edulis and stabilizing them in a range of pH from 7.0 to 9.0. The described method includes the additional steps, after acetic acid extraction of the catechol-containing protein, of removing low molecular weight acid soluble materials from the extract and reacting the extract fraction with a water soluble borate.
Waite, U.S. Pat. No. 4,585,585 discloses methods for the preparation and isolation of decapeptides having the formula ala-lys-pro/hyp-ser/thr-tyr/dopa-pro/hyp-pro/hyp-ser/thr-tyr/dopa-lys and large polyphenolic molecules comprising repeating units of the decapeptide for which the linking groups are amino acids, oligopeptides or bifunctional spacers. Digestion of the polyphenolic proteins in trypsin in the presence of a neutral or slightly basic buffer results in the above decapeptides. The specification states that the decapeptides can be used as building blocks in the construction of larger polyphenolic molecules possessing the adhesive capabilities of the native bioadhesive protein.
When the bioadhesive proteins are treated with clostridial collagenase, the molecular weight is reduced to between about 110,000 and about 130,000. The resultant collagenase-resistant fragments contain most of the HYP and DOPA of the original bioadhesive proteins. Collagenase-resistant fragments are rapidly degraded by trypsin into decapeptides, the repeating unit in the native protein. Alternatively, trypsin digestion may be performed on isolated bioadhesive proteins, without first treating the bioadhesive proteins with clostridial collagenase.
Waite, U.S. Pat. No. 4,687,740 issued from a divisional application of the above U.S. Pat. No. 4,585,585 and relates to the method of preparation of the decapeptide.
A product referred to as Cell-Tak cell and tissue adhesive is available from BioPolymers, Inc. of Farmington,CT and is said to be a highly purified formulation of the adhesive protein of the marine mussel. The use for which it is offered is for cell and tissue culture growth under conditions which are compatible with and encourage cell viability and proliferation.
Since the detection of the adhesive property in the acid-soluble fraction as described in the earlier reports identified above subsequent and recent developments as disclosed in the cited patents, demonstrate that the direction of the field has been toward complete purification, leading to synthetic decapeptides and the synthetic construction of large polyphenolic proteins therefrom. Further, application of these purified and synthetic proteins has been directed toward physiologically favorable conditions, such as those in which not only the cell or tissue culture are to be grown but also the protein itself are not exposed to potentially destructive harsh conditions.